FLJ20420 down-regulates the expression of BAG-1 in lung cancer cell lines.

<p>(A) Luciferase assay with BAG-1 promoter. Luciferase reporter under the control of the BAG-1 promoter was co-transfected in A549 and L9981 cells with either the pcDNA3.1 parent vector or pcDNA3.1-FLJ20420 expression plasmid. Luciferase activity for the BAG-1 promoter is set as 100% upon transfection with the empty pcDNA3.1 vector. Transfection efficiency was determined by co-transfecting the pRL-CMV vector, which encoded the Renilla luciferase gene (0.02 µg). (B) Knockdown of FLJ20420 expression in A549 and L9981 cell lines. Cells were transfected with either FLJ20420 siRNA-1, -2 or scrambled control siRNA. After 48 h of transfection, the cells were collected and total RNA was extracted. FLJ20420 mRNA expression was measured by real-time PCR, in triplicate, as described in the methods section. (C) Microarrays of FLJ20420-silenced A549 and L9981 cells. In both of the FLJ20420-silenced A549 and L9981 cell lines, FLJ20420 expression was dramatically reduced, while BAG-1 expression increased, in comparison to control transfected cells. (D) BAG-1 mRNA expression in FLJ204200-siRNA transfected cells, as measured in triplicate by real-time PCR, and described in the methods section. (E) BAG-1 protein expression in FLJ20420-siRNA transfected cells was detected by Western blotting with anti-BAG-1 antibody at the same time points post-transfection. Tubulin was used as an internal control.